| Mannan-binding lectin (MBL), a member of the collectin superfamily (C-type lectin with a collagen-like domain), is a plasma glycoprotein mainly secreted by liver cells. MBL can form compound with two MBL-associated serine proteases(MASP-1, MASP-2) and then activate the complement cascade via lectin pathway to play the function of anti-infection by innate immunity. Three different genetic polymorphisms in exon 1 independently lead to MBL insufficiency and further to opsonization deficiency. CGT52TGT, GGC54GAC and GGA57GAA mutations result in Arg→Cys, Gly→Asp and Gly→Glu substitutions, respectively. These mutant genes are codominant inheritance on euchromosome. The frequencies of these point mutations vary significantly among ethnics but are very high overall. For instance, the frequency of mutation of codon 54 in exon 1 of MBL gene in Eurasian populations is higher (0.11～0.14), that of codon 57 in sub-Saharan African very high (0.23～0.29), but that of mutation of codon 52 relatively low (≤0.1) in all tested populations. Obviously, MBL insufficiency is one of the most common immunodeficiencies. Recently, more and more data indicate that MBL insufficiency is closely associated with recurrent infection of a wide range pathogen. However, it is still not clear the mechanisms by which the mutations of MBL gene cause immunodeficiency.Previously, our research team transfected the recombinant expression vectors contain wild type and mutant genes into COS7 cells by LipofectamineTM Reagent, respectively. In each transfection, the mass of the expression vectors and the number of COS7 cells were the same. 72 hours later, the supernatants were collected and the expression product in each supernatant was quantitated by ELISA. Analyzed by One-way ANONA, it was found that there were no obvious differencies among the levels of four forms of MBL proteins secreted by COS7 cells. So, we infer that the point mutations in the exon 1 of MBL gene might not interrupt the synthesis and secretion of MBL protein.However, the point mutations in the exon 1 of MBL gene don't interrupt the synthesis and secretion of MBL protein, the reason why MBL protein level reduction in serum—whether is it due to unstable structure or disfunction of MBL protein caused by the mutations of MBL gene? To better understand the molecular mechanism how the mutations of MBL gene lead to immunodeficiency and to further clarify the structure-function relationship of MBL molecule, we need to obtain the stable expression product of the wild type and mutant genes for reseaching their biological behaviors. In the research, we selected the CHO cells contain four eukaryotic recombinant expression vectors (pcDNA4/HisMax C-MBLw, pcDNA4/HisMax C-MBLm52, pcDNA4/HisMax C-MBLm54 and pcDNA4/HisMax C-MBLm57) for one months selection and then obtained the stable and high expression monoclonal cell lines (named CHO-MBLw, CHO-MBLm52, CHO-MBLm54 and CHO-MBLm57) by sandwich ELISA assay. We analyzed the recombinants and research their biological behaviors. The CHO cells transfected blank vector were dealed with equally as a negative control, named CHO-pcDNA4C.1. Estalishment of sandwich ELISAsystemsWe established two sandwich ELISA systems to detect the concentration of the supernatant and select the monoclonal cell lines that express the objective protein stably and efficiently. The first sandwich system: capture antibody was anti-MBL-CRD mAb and the detecting antibody was Biotin-anti-MBL-CRD mAb, for MBL is an oligomer. We suspected it may due to the stereospecific blockade, since the sensibility of this system was only 2.5 mg/L. The second sandwich system: capture antibody was anti-MBL-CRD mAb and the detecting antibody was HRP-anti-HisG. The sensibility of this system was 300μtg/L. The recombinant wild type MBL was used as a standard protein.2. Screening of the CHO cells transfected wild type and variant MBL genes that express the objective proteins stably and efficientlyPreviously, our research team constructed CHO cells that contain respectively four eukaryotic expression vectors: CHO-MBLw, CHO-MBLm52, CHO-MBLm54 and CHO-MBLm57. We resused the cells, monocloned in 96 orifice and selected them by 800 mg/L of Zeocin. The culture solution was changed after 3～5 days to sustain the concentration of Zeocin for 1 months. We obtained respectively 4,1,2,5 monoclonal cell lines and the concentration of the supernatant was 1～2 mg/L by sandwich ELISA assay. The expression of mRNA was analyzed by RT-PCR.3. Study on the characteristics of wild type and variant MBL proteinsThe recombinant protein was purified from the culture supematant by anti-MBL-CRD antibody chromatography and Ni2+-NTA agarose chromatography. We obtained 1 mg recombinant protain in 1L culture supernant. Analyzed by Western blot and ELISA, the purified recombinant proteins, named wild type MBL, 52Cys, 54Asp and 57Glu mutant MBL proteins respectively, can react with anti-MBL-CRD, anti-His, anti-MBL antibodies. Analyzed by SDS-PAGE and Western blot, it was showed that the mutant proteins appear mainly at the site of double strands (Mr 50000) but the wild type protein appears mainly at 2-6 oligomers (Mr 120 000), which indicates that mutant proteins have a much lower oligomerization level than those of the recombinant human wild MBL protein. Analyzed by yeast-coagulation experiment, it was revealed that only wild type and plasma-derived natural can bind with mannan of yeast and form visible coagulating beads, but three mutant MBL proteins form few coagulating beads; It was found by the ligand-binding assay that only protein forms with high oligomerization level, such as wild type or plasma-derived natural MBL, can bind with mannan, but three mutant MBL proteins lose this function due to their low oligomerization. It was demonstrated by complement deposition assay that three mutant MBL proteins can't activate complements through lectin pathway.Doctor Cai xuemin in our team discovered that three mutant MBL proteins depress the ability of binding with MASP by analysis of binding with N terminal fragment of MASR As a result, we concluded that CGT52TGT, GGC54GAC and GGA57GAA point mutations in MBL gene cause the substitution in MBL peptide may disrupt the structure of MBL subunit and the oligomerization, including ligand and MASPs binding ability, as well as complements activation through lectin pathway.
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